Plant Cell Suspension Cultures

Callus Plant Cell Suspension Cultures  Callus  Non-organized tumor tissues  Parenchymatous in nature  Differentiated and non- differentiated cells  Sigmoid pattern of growth during each passage  Cell Suspension Culture A homogenous suspension of dividing cel...

Callus Plant Cell Suspension Cultures  Callus  Non-organized tumor tissues  Parenchymatous in nature  Differentiated and non- differentiated cells  Sigmoid pattern of growth during each passage  Cell Suspension Culture A homogenous suspension of dividing cells Cell suspension culture consists of cell aggregates dispersed and growing in moving liquid media  Uses of Cell Suspension Culture Secondary metabolites production Biotransformation Use of suspension cultures in plant propagation.  Establishment Steps for Cell Suspension Culture 1. Subculture of callus, cell suspension culture 2. Choice of the explants and induction to cell division/ callus formation 3. Inoculum in a liquid culture medium.  Starting Cell Suspension Culture It is usually started by placing an inoculum of friable callus in a liquid medium Placing an ex-plant in liquid culture medium  Leaf Sections Floated in Liquid Culture Medium Leaf sections floated on Murashige and Skoog (1962) medium After several days on a rotary shaker, they can be disintegrated completely to release a great number of cells into suspension.  Disadvantages -Use Explants Floated In Liquid Culture Medium 1. High probability for contamination 2. No formation of callus due to a low gaseous exchange between explants and liquid medium Cell Suspension Culture From Friable Callus Cell suspension culture is normally initiated by transferring pieces of undifferentiated and friable calli to a liquid medium.  Obtaining Friable Callus 1) Choice of ex-plant 2) Identification of a suitable growth medium  Callus As Inoculum In Liquid Culture Medium Callus: Separated from the parent ex-plant and transferred to a fresh medium to build up reasonable amount of callus tissue. Transferred to fresh medium every 4-‐6 weeks. Essential step to avoid cell aging that is visible as reduction of growing and dark spots.  Causes of Explant Aging Nutritive element declining Water loss by evaporation, therefore modification of nutritive compound concentration Build up of compounds products from cell that can have an inhibition of cell growth or induce apoptosis  Callus production from Zygote Embryos of Croton bonplandianum. 1. 10 days old embryo culture 2. One month culture, callus from root (cr) and form endopserm (ce); 3. Healthy callus Size of Callus as Inoculum in Liquid Culture Medium Friable callus in actively division stage of about 5 mm of diamter transferred in a flask (120 ml) containing 30 ml of the suitable liquid medium.  Compact Callus ▪ Compact callus can be an alternative form to friable callus. ▪ Transfer it with part of explant in the liquid culture medium ▪ After a period variable from 7-‐10 days, collect cells and small clumps by a glass pipet t e. ▪ Transfer it in fresh medium.  Media Composition A wide variety of ex-plant and media composition has been used : Heller, B5 Gamborg and MS medium To these media added, vitamins, inositol, sucrose, and auxin (2,4-D) at  1-‐5 M concentrations for cell to divide.  Culture Vessels Wide-‐mounthed Erlemeyer flasks are widely used as culture vessels. The flasks are normally sealed with aluminium foil. Flask closure must maintain sterility, allow gas exchange and reduce evaporation. Cot t on wool plugs may be used for sealing flasks. They are a common source of contamination on flasks that are sit t i n g on a shaker for several weeks.  Orbital Shaker Platform shaker are widely used for the initiation and serial propagation of plant cell suspension culture. They should have a variable speed control (30-‐120 rpm). The shaker should be kept in the air-‐conditioned room with good temperature control.  Agitation of medium serves two purposes 1. It exerts a mild pressure on cell aggregates, breaking them into smaller clumps and single cells. 2. It maintains uniform distribution of cell and cell clumps in the medium. 3. Movement of the medium also provides good gaseous exchange between the culture medium and air.  Fine Cell Suspension Culture In an ideal cell suspension culture, there are single isodiametric cells and few clumps of 20-‐100 cells. Mostly of cell suspension culture is made up by heterogeneous cell population either by size and by specific density. Cell Suspension Culture Completely Isolated Cells Because the walls of plant cells have a natural tendency to adhere, it is not possible to obtain suspensions that consist only of dispersed single cells. Some progress has been made in selecting cell lines with increased cell separation  Cell Proportion and Size The proportion and size of small cell aggregates varies according to plant variety and the medium in which the culture is grown. As cells tend to divide more frequently in aggregates than in isolation, the size of cell clusters increases during the phase of rapid cell division. Use of Cell Density for Obtaining Fine Cell Suspension Culture Cells can be separated according to their size, shape, density, medium viscosity by centrifugation Type of Hormones Influence Degree of Cell Dispersion The degree of cell dispersion in suspension cultures is particularly influenced by the concentration of growth regulators in the culture medium. Auxinic growth regulators: increase the specific activity of enzymes, which bring about the dissolution of the plant cell wall  Effects of PGRs on Cell Suspension Aggregates Auxin at relatively high concentration and a low concentration of a cytokinin in a liquid culture medium usually increase cell dispersion. Use of high auxin levels or low auxin/ low cytokinin to obtain maximum cell dispersion will ensure that the cultured cells remain undifferentiated. Establishment Steps for Cell Suspension Culture 1. Choice of the explant and induction to cell division 2. Inocolum in a liquid culture medium. 3. Subculturing of cell suspension culture  Subculturing Cell suspension culture must be frequently and on regular bases transfer in a fresh medium. The lag period is usually between one or two weeks. The ratio of dilution (cell vs medium) is experimentally determined- As general rule: 1: 4 after one week 1:10 after two weeks Sterility of Cell Suspension Culture The sterility of plant cell suspension can be monitored by several parameters: – Change in colour of solution – Change in pH – Smell – Use of microscope Establishment of Cell Culture  Callus Cultures The most common growth parameters used for callus cultures include; Fresh weight Dry weight Growth index Dry-Weight Determination (Callus) It can be estimated by drying the tissue at 60°C until of constant weight (usually 12 -16 h). 1. Take a sample of the fresh tissue, weigh it on a pre-‐weighed square of aluminum foil, and evaporate the water contained in the tissue in the pre-‐heated oven at 60°C for 12 h. 2. Allow samples to cool down in a dessicator containing silica gel for 15–20 min and then register the sample’s weight. Dry-Weight Determination (Callus) 3. Put the tissue sample back into the oven and take a new weight register after 2 h. If no variations are detected, samples have reached constant weight. 4. If variations higher than 10% regarding the previous register observed, return samples to the oven for another 2-‐h period. 5. Alternatively, dry weight can be obtained from lyophilized tissues. Once harvested, fresh tissues should be weighed, deposited in lyophilizer flasks, and frozen at –20°C for at least 12 h. 6. Flasks with frozen tissues are then connected to the vacuum line for 24 h and weight registered  Monitoring Cell Suspension Culture Cell Cell Vitality Growth